Purification of cystathionine beta-synthase

ABSTRACT

This invention provides chromatographic methods for the purification of a cystathionine β-Synthase (CBS) protein, particularly truncated variants thereof and compositions and pharmaceutical compositions prepared therefrom.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/178,024 filed Nov. 1, 2018; which is a continuation of U.S. patentapplication Ser. No. 14/970,814 filed Dec. 16, 2015 (issued as U.S. Pat.No. 10,160,962); which is a continuation of U.S. patent application Ser.No. 13/830,494 (issued as U.S. Pat. No. 9,243,239) filed Mar. 14, 2013;which claims priority to U.S. provisional application 61/615,629 filedMar. 26, 2012; the disclosure of each of which is hereby incorporated byreference in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing file, entitledSEQLST_2089-1002USCON3.txt, was created on Jan. 7, 2020, and is 13,242bytes in size. The information in electronic format of the SequenceListing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to methods for purification ofCystathionine β-Synthase (CBS), particularly truncated variants thereof.The present invention also relates to compositions of substantially pureCBS produced through said methods of purification.

BACKGROUND OF THE INVENTION

Cystathionine β-synthase (CBS) plays an essential role in homocysteine(Hcy) metabolism in eukaryotes (Mudd et al., 2001, in The Metabolic andMolecular Bases of Inherited Disease, 8 Ed., pp. 2007-2056, McGraw-Hill,New York). The CBS enzyme catalyzes a pyridoxal 5′-phosphate (PLP;Vitamin B₆)-dependent condensation of serine and homocysteine to formcystathionine, which is then used to produce cysteine by anotherPLP-dependent enzyme, cystathionine γ-lyase. In mammalian cells thatpossess the transsulfuration pathway, CBS occupies a key regulatoryposition between the remethylation of Hcy to methionine or itsalternative use in the biosynthesis of cysteine. The relative fluxbetween these two competing pathways is roughly equal and is controlledby intracellular S-adenosylmethionine (AdoMet) concentrations(Finkelstein and Martin, 1984, J. Biol. Chem. 259:9508-13). AdoMetactivates the mammalian CBS enzyme by as much as 5-fold with an apparentdissociation constant of 15 μM (Finkelstein et al., 1975, Biochem.Biophys. Res. Commun. 66: 81-87; Roper et al., 1992, Arch. Biochem.Biophys. 298: 514-521; Kozich et al., 1992, Hum. Mutation 1: 113-123).

The C-terminal regulatory domain of human CBS consists of ˜140 aminoacid residues (Kery et al., 1998, Arch. Biochem. Biophys. 355: 222-232).This region is required for tetramerization of the human enzyme andAdoMet activation (Kery et al., 1998, id.). The C-terminal regulatoryregion also encompasses the previously defined “CBS domains” (Bateman,1997, Trends Biochem. Sci. 22: 12-13). These hydrophobic sequences (CBS1 and CBS 2), spanning amino acid residues 416-468 and 486-543 of SEQ IDNO: 1, respectively, are conserved in a wide range of otherwiseunrelated proteins. Their function remains unknown, although the sharptransition of thermally induced CBS activation and the observation thatmutations in this domain can constitutively activate the enzymeindicates that they play a role in the autoinhibitory function of theC-terminal region (Janosik et al., 2001, Biochemistry 40: 10625-33; Shanet al., 2001, Hum. Mol. Genet. 10: 635-643; Miles and Kraus, 2004, J.Biol. Chem. 279: 29871-4). Two well-conserved CBS domains are alsopresent in the C-terminal region of the yeast CBS, which is ofapproximately the same length as the human enzyme.

In healthy normal individuals, CBS-mediated conversion of Hcy tocystathionine is the rate-limiting intermediate step of methionine (Met)metabolism to cysteine (Cys). Vitamin B₆ is an essential coenzyme forthis process. In patients with certain genetic mutations in the CBSenzyme, the conversion of Hcy to cystathionine is slowed or absent,resulting in elevations in the serum concentrations of the enzymaticsubstrate (Hcy) and a corresponding decrease in the serum concentrationsof the enzymatic product (cystathionine). The clinical condition of anelevated serum level of Hcy, and its concomitant excretion into theurine, is collectively known as homocystinuria.

Deficiency of CBS is the most common cause of inherited homocystinuria,a serious life-threatening disease that results in severely elevatedhomocysteine levels in plasma, tissues and urine. Estimates on theprevalence of homocystinuria vary widely. Ascertainment by newbornscreening and clinical ascertainment have indicated a prevalence rangingfrom 1:200,000 to 1:335,000 (Mudd et al., 1995, The Metabolic andMolecular Basis of Inherited Diseases, McGraw-Hill: New York, p. 1279).The primary health problems associated with CBS-deficient homocystinuria(CBSDH) include: cardiovascular disease with a predisposition tothrombosis, resulting in a high rate of mortality in untreated andpartially treated patients; connective tissue problems affecting theocular system with progressive myopia and lens dislocation; connectivetissue problems affecting the skeleton characterized by marfanoidhabitus, osteoporosis, and scoliosis; and central nervous systemproblems, including mental retardation and seizures. Symptoms includedislocated optic lenses, skeletal disorders, mental retardation andpremature arteriosclerosis and thrombosis (Mudd et al., 2001, id.).Homozygous CBS deficiency is associated with a multitude of clinicalsymptoms, including mental retardation, osteoporosis, kyphoscoliosis,stroke, myocardial infarction, ectopia lentis, and pulmonary embolism.Cardiovascular complications of the disease, in particular arterial andvenous thrombosis, are the principal contributors to early mortality.

The pathophysiology of CBS deficiency is undoubtedly complex, but thereis a consensus that the fundamental instigator of end-organ injury is anextreme elevation of serum Hcy, a substrate of CBS that builds-up intissues and blood due to the absence of its CBS-catalyzed condensationwith L-serine to form cystathionine. The toxicity of profound elevationsin blood and tissue concentrations of Hcy may ensue from the molecularreactivity and biological effects of Hcy per se or from its metabolites(e.g. Hcy-thiolactone) that affect a number of biological processes(Jakubowski et al., 2008, FASEB J 22: 4071-6). Abnormalities in chronicplatelet aggregation, changes in vascular parameters, and endothelialdysfunction have all been described in patients with homocystinuria.

Currently, three treatment options exist for the treatment of CBSDH:

-   -   1) Increase of residual activity of CBS activity using        pharmacologic doses of Vitamin B₆ in Vitamin B₆-responsive        patients    -   2) Lowering of serum Hcy by a diet with a strict restriction of        the intake of Met; and    -   3) Detoxification by betaine-mediated conversion of Hcy into        Met, thus lowering serum Hcy concentration.

Each of these three therapies is aimed at lowering serum Hcyconcentration. The standard treatment for individuals affected withVitamin B₆ non-responsive CBSDH consists of a Met-restricted dietsupplemented with a metabolic formula and Cys in the form of cysteine(which has become a conditionally essential amino acid in thiscondition). Intake of meat, dairy products, and other food high innatural protein is prohibited. Daily consumption of a poorly palatable,synthetic metabolic formula containing amino acids and micronutrients isrequired to prevent secondary malnutrition. Supplementation with betaine(trade name: Cystadane™, synonym: trimethylglycine) is also standardtherapy, wherein betaine serves as a methyl donor for the remethylationof Hcy to Met catalyzed by betaine-homocysteine methyltransferase in theliver (Wilcken et al., 1983, N. Engl. J. Med. 309: 448-53). Dietarycompliance generally has been poor, even in those medical centers whereoptimal care and resources are provided, and this non-compliance hasmajor implications on the development of life-threatening complicationsof homocystinuria.

To enable patients with homocystinuria enjoy a far less restrictive diet(e.g. daily intake limited to 2 g protein per kg, which is easilyattainable), and have a significantly decreased Hcy plasma level leadingin the long-term to clinical improvement, a strategy for increasingenzyme activity provides potential for treatment as set forth inco-pending U.S. provisional patent application Ser. No. 61/758,138. Themost effective therapeutic strategy is to increase enzyme activity, asis evident when Vitamin B₆-responsive homocystinuria patients are givenpyridoxine. However, this strategy is not possible for Vitamin B₆non-responsive patients due to the nature of the mutations. Enzymereplacement therapy (ERT) as a way to increase enzyme activity in thesepatients requires exogenous enzyme, which is not present in the art andthus raises a need in the art for improved reagents and methods forproducing CBS in greater yields of sufficiently purified enzyme fortherapeutic administration.

Kraus and colleagues have developed expression systems and fermentationconditions for generating active recombinant human CBS and variantsthereof (U.S. Pat. Nos. 5,635,375, 5,523,225 and 7,485,307, incorporatedby reference herein in their entireties for any purpose). These proteinswere purified by processes relevant for academic purposes, including useof protein leads on the proteins which are not considered useful forpreparation of pharmaceuticals.

In order to employ methods of increasing CBS enzyme activity, anefficient method of CBS enzyme purification is required. Existingmethods of purification for recombinant CBS protein rely on affinitytags to facilitate purification that does not provide the desired purityand efficiency. Therefore to more efficiently obtain the necessarylevels of CBS required for therapeutic use there is a need for improveddownstream purification of CBS protein produced in microbial cells.

SUMMARY OF THE INVENTION

This invention provides methods for purifying cystathionine β-Synthase(CBS), wherein said CBS protein is a naturally occurring truncatedvariant, or a chemically cleaved or genetically engineered truncatethereof, and particularly truncated CBS produced in recombinant cells.In particular embodiments, the method comprises the steps of: (a)providing a CBS-containing solution in the presence of at least oneimpurity; and (b) performing chromatographic separation of saidCBS-containing solution using a metal affinity chromatography (IMAC)resin. In additional particular embodiments, the method comprises thesteps of: (a) providing a CBS-containing solution in the presence of atleast one impurity; and (b) performing chromatographic separation ofsaid CBS-containing solution using an ion exchange chromatography columnand a metal affinity chromatography (IMAC) resin.

In certain embodiments the method further comprises performance ofadditional chromatographic steps (known in the art as “polishing”steps). In particular embodiments, the methods of the invention includethe step of performing chromatographic separation using a HydrophobicInteraction Chromatography (HIC) column. In other embodiments the methodfurther comprises the step of performing chromatographic separationusing a ceramic hydroxyapatite resin.

In certain embodiments the ion exchange column is an anion exchanger,preferably a weak anion exchanger. In particular embodiments the anionexchanger is a DEAE-Sepharose FF column. In further embodiments the IMACresin is charged with a divalent ion. In yet further embodiments thedivalent metal ion is nickel, copper, cobalt or zinc. In more specificembodiments the divalent metal ion is zinc.

In certain other embodiments the method further comprises eluting CBSfrom the IMAC resin with an elution buffer comprising imidazole. Incertain embodiments the CBS-containing solution is a clarified CBSsolution, wherein cell debris and other particulate matter is removedfrom a suspension comprising CBS including but not limited tosupernatant after centrifugation or filtrate after filtration. In yetother embodiments the CBS-containing solution is obtained byhomogenizing cells expressing a recombinant construct comprising anucleic acid sequence encoding CBS. In certain embodiments the CBSnucleic acid sequence comprises SEQ ID NO. 1 and encodes a protein havethe amino acid sequence identified as SEQ ID NO: 2. In other embodimentsthe nucleic acid sequence is truncated. In yet other embodiments thetruncated CBS nucleic acid sequence has been truncated to an endingposition of one of amino acid residues from 382-532, 382-550 or 543-550of SEQ ID NO:2

In other certain embodiments the recombinant cells are microbial cells,particularly bacterial cells. In particular embodiments, the bacterialcells are E. coli cells, particularly recombinant E. coli cells thatproduce a mammalian, preferably human, CBS protein. In certainparticular embodiments, said human CBS protein has an amino acidsequence as set forth in SEQ ID NO:3 or a truncated CBS nucleic acidsequence that has been truncated to an ending position of one of aminoacid residues from 382-532 or 543-550 of SEQ ID NO:2. In otherparticular embodiments, the truncated CBS nucleic acid sequence isoptimized for expression in E. coli, identified by SEQ ID NO: 4.

In another aspect, a substantially purified CBS solution is providedusing a method comprising the steps of: a) providing a CBS-containingsolution in the presence of at least one impurity, wherein said CBSprotein is a naturally occurring truncated variant, or a chemicallycleaved or genetically engineered truncate thereof, and particularlytruncated; and (b) performing chromatographic separation of saidCBS-containing solution using a metal affinity chromatography (IMAC)resin. In additional particular embodiments, a substantially purifiedCBS solution is provided using a method comprising the steps of: (a)providing a CBS-containing solution in the presence of at least oneimpurity; and (b) performing chromatographic separation of saidCBS-containing solution using an ion exchange chromatography column anda metal affinity chromatography (IMAC) resin.

In certain embodiments of the invention the substantially purified CBSsolution is formulated in a pharmaceutically acceptable carrier.

In another aspect, the invention provides methods for producing anenriched CBS solution, the method comprising of: (a) providing aCBS-containing solution in the presence of at least one impurity,wherein said CBS protein is a naturally occurring truncated variant, ora chemically cleaved or genetically engineered truncate thereof, andparticularly truncated; and (b) performing chromatographic separation ofsaid CBS-containing solution using an immobilized metal affinitychromatography (IMAC) resin charged with a divalent metal ion.

In another aspect, an enriched CBS solution is provided using a methodcomprising the steps of: a) providing a CBS-containing solution in thepresence of at least one impurity, wherein said CBS protein is anaturally occurring truncated variant, or a chemically cleaved orgenetically engineered truncate thereof, and particularly truncated; and(b) performing chromatographic separation of said CBS-containingsolution using an immobilized metal affinity chromatography (IMAC) resincharged with a divalent metal ion.

It is a particular advantage of this invention that purification ofrecombinant, full-length or truncated CBS, particularly human CBS, canbe achieved without further modification of the protein, e.g., byincorporating a “tag” molecule known in the art (poly-HIS, FLAG, etc.).Use of the chromatographic methods disclosed herein advantageously makesthese tags unnecessary, thus avoiding additional recombinantmanipulation and any disadvantages (in immunogenicity, in vivo half-lifeor biochemical activity) that might be introduced into any preparationof recombinant CBS containing such a tag.

Specific preferred embodiments of the invention will become evident fromthe following more detailed description of certain preferred embodimentsand the claims.

BRIEF DESCRIPTION OF DRAWINGS

The following detailed description of the embodiments of the presentinvention can be best understood when read in conjunction with thefollowing drawings.

FIG. 1 is a purification train summary from scale-up generation runsusing a multi-step chromatography method including DEAE-Sepharose-FF,Zn-IMAC and HIC chromatography.

FIG. 2 is a purification summary from purification experiments using aDEAE-Sepharose-FF column and CBS purified using the “non-optimized”bacterial expression construct. Mobile phases included 10% ethyleneglycol in addition to other components as set forth in the Examples.

FIG. 3 is a purification train summary from scale-up generation runsusing a multi-step chromatography method including DEAE-Sepharose-FF,Zn-IMAC, ceramic hydroxyapatite resin and HIC chromatography.

FIG. 4 is a photoimage of a SDS page gel showing the relative amounts ofCBS protein and impurities for each stage of the purification step usinga DEAE column.

FIG. 5 is a photoimage of a SDS page gel showing the relative amounts ofCBS protein and impurities for a 3 column purification method including:a DEAE column, a Zn-IMAC column and HIC column.

FIG. 6 is a photoimage of a SDS page gel showing the relative amounts ofCBS protein and impurities for a 4 column purification method including:a DEAE column, a Zn-IMAC column, a ceramic hydroxyapatite resin and aHIC column.

FIG. 7 is chromatograms demonstrating the components of the separatedmixture following purification using Zn-IMAC.

FIG. 8 is a purification summary from development runs using a Ni-IMACcolumn.

FIG. 9 is a summary table demonstrating the total protein following apurification method using a Ni-IMAC column.

FIG. 10 is a photoimage of a SDS page gel showing the relative amountsof CBS protein and impurities for each stage of the purification stepusing a Ni-IMAC column.

FIG. 11 is a purification summary from scale-up generation runs using aCu-IMAC column.

FIG. 12 is a summary table demonstrating the total protein following apurification method using a Zn-IMAC column.

FIG. 13 is a photoimage of a SDS page gel showing the relative amountsof CBS protein and impurities for each stage of the purification stepusing a Zn-IMAC column.

FIG. 14 is a scheme of the purification method using multi-stepchromatography purification steps.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides methods for purification of CBS protein, whereinsaid CBS protein is a naturally occurring truncated variant, or achemically cleaved or genetically engineered truncate thereof, andparticularly a truncated protein CBS produced in recombinant cells. Inparticular, the invention provides methods for the purification of a CBSprotein that include the steps (a) providing a CBS-containing solutionin the presence of at least one impurity; and (b) performingchromatographic separation of said CBS-containing solution using a metalaffinity chromatography (IMAC) resin. In additional particularembodiments, the method comprises the steps of: (a) providing aCBS-containing solution in the presence of at least one impurity; and(b) performing chromatographic separation of said CBS-containingsolution using an ion exchange chromatography column and a metalaffinity chromatography (IMAC) resin.

A particular chromatographic separation step in the certain embodimentsof the methods provided by this invention comprises an ion exchangechromatography column. In one embodiment, the ion exchangechromatography column is an anion exchanger, preferably a weak anionexchanger. Various types of anion exchange resins can be used, includingDEAE-Sephadex, QAE-Sephadex, DEAE-Sephacel, DEAE-cellulose andDEAE-Sepharose-FF. According to one embodiment, the anion exchange resinis DEAE-Sepharose-FF.

Another particular chromatographic separation step in the certain of themethods provided by this invention comprises a metal affinitychromatography (IMAC) resin having appropriate pH and conductivity suchto allow the protein to bind to the column while selective intermediatewashes are used to remove weaker binding proteins and other molecularspecies. In certain embodiments, varying concentrations of imidazolewere used to modulate the partitioning during the chromatography.Suitable metal affinity resins include immobilized metal affinitycolumns charged with a divalent metal ion including nickel, copper,cobalt or zinc. In certain embodiments of the methods of the invention,the metal affinity chromatography (IMAC) column is used following ionexchange chromatography. In such embodiments, the IMAC column ispreferably charge with zinc as a divalent cation. In other embodimentsof the inventive methods, the IMAC column is used as an initialchromatographic step. In such embodiments, nickel or copper divalentcations are preferably used to charge the IMAC column.

Additional chromatographic steps provided in certain embodiments of themethods of this invention for purifying CBS from a CBS-containingsolution include without limitation hydrophobic interactionchromatography (HIC). HIC is useful for removing impurities that haverelatively closely related chromatographic properties that are elutedtogether with the target protein during the capture step.

Further additional chromatographic steps provided in certain embodimentsof the methods of this invention for purifying CBS from a CBS-containingsolution include without limitation a ceramic hydroxyapatite resin.“Ceramic hydroxyapatite” or “CHAP” refers to an insoluble hydroxylatedcalcium phosphate of the formula (Ca₁₀(PO₄)₆(OH)₂), which has beensintered at high temperatures into a spherical, macroporous ceramicform. The methods of the invention also can be used with hydroxyapatiteresin that is loose or packed in a column The choice of columndimensions can be determined by the skilled artisan.

Chromatography matrices useful in the method of the invention arematerials capable of binding biochemical compounds, preferably proteins,nucleic acids, and/or endotoxins, wherein the affinity of saidbiochemical compounds to said chromatography matrix is influenced by theion composition of the surrounding solution (buffer). Controlling theion composition of said solution allows to use the chromatographymaterials of the invention either in subtractive mode (CBS passesthrough said chromatography matrix, at least certain contaminants bindto said chromatography matrix) or, preferably, in adsorptive mode (CBSbinds to the chromatography matrix).

In particular embodiments, the method for purification comprises thestep of homogenizing host cells, particularly recombinant cells and incertain embodiments, recombinant cells producing mammalian, preferablehuman, CBS protein, wherein said recombinant construct encodes a CBSprotein that is a naturally occurring truncated variant, or agenetically engineered truncate thereof, and particularly wherein saidconstruct has been optimized for recombinant cell expression. Inparticular embodiments, said recombinant cells are microbial cells andparticularly bacterial cells. In certain particular embodiments, thebacterial cells are E. coli cells and the CBS sequence has beenengineered in the recombinant expression construct to be optimized forexpression in said cells; a specific embodiment of such a nucleic acidsequence optimized for CBS expression in E. coli is set forth in SEQ IDNO: 4. In said methods, cells are harvested, e.g. by centrifugation, andoptionally stored at −80 degree ° C. Homogenization of host cells isperformed by disrupting the cells host using physical, chemical orenzymatic means or by a combination thereof. Advantageously, forpurification from bacterial sources homogenation is performed bydisrupting the cell wall of said bacterial host by sonication.Alternatively or additionally homogenizing is performed by destabilizingthe bacterial cell wall of the host by exposure to a cell wall degradingenzyme such as lysozyme.

The methods of the invention can further comprise a clarified CBShomogenate, wherein cell debris is removed from the homogenate by eitherfiltration or centrifugation. In certain embodiments, clarifying isperformed by centrifuging the homogenate at an effective rotationalspeed. The required centrifugation time depends inter alia on the volumeof the homogenate, which can be determined empirically to obtain asufficiently solid pellet. To obtain an essentially cell debris-freeclarified homogenate a combination of centrifugation and filtration canbe performed on the homogenate.

The term “recombinant cell” as used herein refers to suitable cells(including progeny of such cells) from any species into which has beenintroduced a recombinant expression construct capable of expressing anucleic acid encoding CBS protein, preferably human CBS protein and mostparticularly a human CBS protein that is a naturally occurring truncatedvariant, or a chemically cleaved or genetically engineered truncatethereof. In specific embodiments, the truncated CBS protein encoded bysaid recombinant expression construct has an amino acid sequence as setforth in SEQ ID NO: 3.

The term, “bacterial cell”, as used herein refers to bacteria thatproduces a mammalian, preferably human, CBS protein inter alia usingrecombinant genetic methods including progeny of said recombinant cell,wherein said CBS protein is a naturally occurring truncated variant, ora genetically engineered truncate thereof.

The term “recombinant expression construct” as used herein refers to anucleic acid having a nucleotide sequence of a mammalian, preferablyhuman, CBS protein, and sequences sufficient to direct the synthesis ofCBS protein in cultures of cells into which the recombinant expressionconstruct is introduced and the progeny thereof.

As used herein, reference to CBS protein or polypeptide preferablyincludes a naturally occurring truncated variant, or a chemicallycleaved or genetically engineered truncate thereof, or fusion proteins,or any homologue (variant, mutant) thereof, and specifically mammalianCBS and preferably human CBS. Such a CBS protein can include, but is notlimited to, purified CBS protein, recombinantly produced CBS protein,soluble CBS protein, insoluble CBS protein, and isolated CBS proteinassociated with other proteins. In addition, a “human CBS protein”refers to a CBS protein from a human (Homo sapiens) preferably includesa naturally occurring truncated variant, or a chemically cleaved orgenetically engineered truncate thereof. As such, a human CBS proteincan include purified, partially purified, recombinant, mutated/modifiedand synthetic proteins. As disclosed herein and in related U.S. Pat.Nos. 8,007,787 and 7,485,307, the CBS protein truncates areadvantageously soluble CBS proteins that are produced in bacteriawithout the creation of insoluble inclusion bodies.

As used herein, the term “homologue” (or variant or mutant) is used torefer to a protein or peptide which differs from a naturally occurringprotein or peptide (i.e., the “prototype” or “wild-type” protein) bymodifications to the naturally occurring protein or peptide, but whichmaintains the basic protein and side chain structure of the naturallyoccurring form. Such changes include, but are not limited to: changes inone, few, or even several amino acid side chains; changes in one, few orseveral amino acids, including deletions (e.g., a truncated version ofthe protein or peptide), insertions and/or substitutions; changes instereochemistry of one or a few atoms; and/or minor derivatizations,including but not limited to: methylation, glycosylation,phosphorylation, acetylation, myristoylation, prenylation, palmitation,amidation and/or addition of glycosylphosphatidyl inositol. A homologuecan have enhanced, decreased, changed, or substantially similarproperties as compared to the naturally occurring protein or peptide. Ahomologue can include an agonist of a protein or an antagonist of aprotein.

Homologues can be the result of natural allelic variation or naturalmutation. A naturally occurring allelic variant of a nucleic acidencoding a protein is a gene that occurs at essentially the same locus(or loci) in the genome as the gene which encodes such protein, butwhich, due to natural variations caused by, for example, mutation orrecombination, has a similar but not identical sequence. Allelicvariants typically encode proteins having similar activity to that ofthe protein encoded by the gene to which they are being compared. Oneclass of allelic variants can encode the same protein but have differentnucleic acid sequences due to the degeneracy of the genetic code.Allelic variants can also comprise alterations in the 5′ or 3′untranslated regions of the gene (e.g., in regulatory control regions).Allelic variants are well known to those skilled in the art.

Homologues can be produced using techniques known in the art for theproduction of proteins including, but not limited to, directmodifications to the isolated, naturally occurring protein, directprotein synthesis, or modifications to the nucleic acid sequenceencoding the protein using, for example, classic or recombinant DNAtechniques to effect random or targeted mutagenesis. CBS variants aredescribed in U.S. Pat. No. 8,007,787, which is incorporated herein byreference in its entirety; in particular and preferred embodiments, thereagents and methods of the invention set forth herein preferablyinclude a naturally occurring truncated variant, or a chemically cleavedor genetically engineered truncate of human CBS protein. Particulartruncated forms of SEQ ID NO: 3 according to the present inventioninclude N-terminal deletion variants, C-terminal deletion variants, andvariants having both N-terminal and C-terminal deletions.

As used herein, “substantially pure” refers to a purity that allows forthe effective use of the protein in vitro, ex vivo or in vivo. For aprotein to be useful in vitro, ex vivo or in vivo, it is preferablysubstantially free of contaminants, other proteins and/or chemicals thatmight interfere or that would interfere with its use, or that at leastwould be undesirable for inclusion with a CBS protein (includinghomologues thereof).

As used herein an enriched CBS solution is a solution subjected to oneor more purification steps.

The purity of protein can be determined by calculating foldpurification, i.e. a formula that provides a measure of how much more apurified solution is compared to a less purified solution or crudeextract. Fold purification is calculated using the following formula:

Specific Activity Final Fraction/Specific Activity Crude Fraction—

Another measurement to assess purity is the “specific activity” whichmeasures the purity of an enzyme. Specific activity can be measuredusing the following formula:

${\frac{Units}{mL} \times \frac{mL}{mg}} = \frac{Units}{mg}$

CBS protein compositions provided by this invention are useful forregulating biological processes and particularly, processes associatedwith the catalysis of the pyridoxal 5′-phosphate (PLP)-dependentcondensation of serine and homocysteine to form cystathionine. Inparticular, compositions of the present invention are useful forproducing cystathionine and cysteine in vitro or for treating a patientthat will benefit from increased CBS activity (e.g., a patient withhomocystinuria). In certain embodiments, the invention provides saidcompositions of CBS protein, preferably human CBS protein, wherein saidCBS protein is a naturally occurring truncated variant, or a chemicallycleaved or genetically engineered truncate of human CBS protein, aspharmaceutical compositions comprising said CBS protein and apharmaceutically acceptable carrier.

As used herein, a “pharmaceutically acceptable carrier” includespharmaceutically acceptable excipients and/or pharmaceuticallyacceptable delivery vehicles, suitable for use in suitableadministration of the composition in vitro, ex vivo or in vivo. Suitablein vitro, in vivo or ex vivo administration preferably comprises anysite where it is desirable to regulate CBS activity. Suitablepharmaceutically acceptable carriers are capable of maintaining a CBSprotein as provided by this invention in a form that, upon arrival ofthe protein at the target cell or tissue in a culture or in patient, theprotein has its expected or desired biological activity. Examples ofpharmaceutically acceptable carriers include, but are not limited towater, phosphate buffered saline, Ringer's solution, dextrose solution,serum-containing solutions, Hank's solution, other aqueousphysiologically balanced solutions, oils, esters and glycols. Aqueouscarriers can contain suitable auxiliary substances required toapproximate the physiological conditions of the recipient, for example,by enhancing chemical stability and isotonicity. Compositions of thepresent invention can be sterilized by conventional methods and/orlyophilized.

Each reference described and/or cited herein is incorporated byreference in its entirety.

The following examples are provided for the purpose of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Example 1. Production of Truncated CBS Protein in Bacteria

A truncated human CBS variant lacking specific portions of thenon-conserved regions (r-hCβSΔC; SEQ ID No: 3) were constructed andover-expressed using the previously described E. coli based expressionsystem (Kozich and Kraus, 1992, supra). In the modification of thissystem disclosed herein (i.e., expressing the truncate rather than thefull-length CBS protein), the CBS truncate encoded by SEQ ID NO: 3 wasexpressed without any fusion partner under the control of the tacpromoter. Constructs encoding the truncated human CBS protein variantr-hCβSΔC (SEQ ID NO: 4) were generated by a modification of thepreviously described pHCS3 CBS expression construct (Kozich and Kraus,1992, Hum. Mutat. 1, 113-123) which contains the CBS full-length codingsequence (SEQ ID NO: 1) cloned into pKK388.1. In this construct, CBSexpression was governed by the IPTG inducible lac promoter. To generateC-terminal deletion constructs, CBS cDNA fragments spanning the desirednucleotide residues were amplified using primers incorporating Sph I andKpn I sites to the 5′ and 3′ respective ends of the PCR product. All PCRproducts were then cut with Sph I and Kpn I and cloned by ligation intothe pHCS3 vector digested with Sph I and Kpn I. An Sph I site naturallyoccurs in the CBS cDNA, just upstream of the antisense primerhybridization site (base pare position 1012, according to the CBS cDNAnumbering, ref. 25). PCR products thus generated were then digested withNco I and Sph I and ligated into the pHCS3 plasmid cut with the sameenzymes.

pKK CBS Δ414-551 sense: (SEQ ID NO: 5)CGTAGAATTCACCTTTGCCCGCATGCTGAT (SphI) antisense: (SEQ ID NO: 6)TACGGGTACCTCAACGGAGGTGCCACCACCAGGGC (KpnI)

Finally, the construct was transformed into E. coli BL21 (Stratagene).The authenticity of the construct was verified by DNA sequencing using aThermo Sequenase Cy5.5 sequencing kit (Amersham Pharmacia Biotech) andthe Visible Genetics Long-Read Tower System-V3.1 DNA sequencer accordingto the manufacturer's instructions.

Bacterial Expression Analysis of CBS Deletion Mutants and Growth of E.coli—

BL21 cells bearing the CBS truncation mutant construct, induction ofexpression and the generation of crude cell lysates were performed asdescribed previously (Maclean et al., 2002, Hum. Mutat. 19(6), 641-55).Briefly, bacteria were grown at 37° C. aerobically in 1 L NZCYMT media(Gibco/BRL, Gaithersburg, Md.) containing 75 μg/mL ampicillin and 0.001%thiamine in the presence or absence of 0.3 mM δ-aminolevulinate (δ-ALA)until they reached turbidity of 0.5 at 600 nm. IPTG was then added to0.5 mM and the bacteria were grown further. The insoluble fraction wasprepared as follows: after the centrifugation of the sonicatedhomogenate, pelleted cell debris were thoroughly washed with chilled IxTris-buffered saline, pH 8.0. The pellets were then resuspended in 1 mlof the lysis buffer (Maclean et al., ibid.) followed by a briefsonication in order to homogenize the insoluble fraction.

CBS Activity Assay—

CBS activity was determined by a previously described radioisotope assayusing [¹⁴C] serine as the labeled substrate (Kraus, 1987, MethodsEnzymol. 143, 388-394). Protein concentrations were determined by theLowry procedure (Lowry et al., 1951, J. Biol. Chem. 193, 265-275) usingbovine serum albumin (BSA) as a standard. One unit of activity isdefined as the amount of CBS that catalyzes the formation of 1 μmol ofcystathionine in 1 h at 37° C.

Denaturing and Native Polyacrylamide Gel Electrophoresis and WesternBlotting—

Western blot analysis of crude cell lysates under both denaturing andnative conditions was performed as described previously (Janosik, 2001,supra) with some modifications. Soluble fractions of E. coli lysatescontaining the expressed mutant protein were mixed with sample bufferand run on a 6% native PAGE without a stacking gel. The finalcomposition of the sample buffer was: 50 mM Tris-HCl, pH 8.9, 1 mM DTT,10% glycerol, 0.001% bromophenol blue. Detection of heme was performedusing a previously described method that relies on heme peroxidaseactivity (Vargas et al., 1993, Anal. Biochem. 209(2), 323-6).

Densitometric Scanning Analysis—

Quantitative densitometry analysis was performed using the ImagemasterID (version 2.0) software (Pharmacia). To construct a calibration curve,50, 75, 100, 250, 500 and 1000 ng of purified wild type CBS protein wererun on an SDS-PAGE together with crude cell lysates of the individualmutants. Following electrophoresis, Western blot immunoanalysis wasconducted using rabbit anti-CBS serum. The signals corresponding to theexperimentally observed CBS mutant subunits were all within the linearrange of the calibration curve constructed with purified human CBS.

Example 2. Preparation of Crude Extraction

Crude CBS protein-containing extracts was prepared for use in downstreamchromatography steps. Frozen pellets (cells) obtained from fermentationof recombinant bacteria producing human truncated CBS variant (r-hCβSΔC;SEQ ID No: 3) were lysed, wherein said bacteria expressed truncatedhuman CBS encoded by SEQ ID NO: 4. Lysis buffer for initial isolationscontained 1 mM DTT, 1% Triton X-100, and Protease Inhibitor. Thesecomponents were eventually removed from the buffer. The buffer used forthe final isolations that produced material for scale-up runs consistedof 20 mM Sodium Phosphate, 50 mM NaCl, 0.1 mM PLP (pH 7.2), withlysozyme added to a concentration of 2 mg/mL after homogenization.Following mixing with lysozyme for 1 hr at 4° C., the homogenate wassonicated until viscosity was reduced and then subjected tocentrifugation at 20,000 rpm (48,000×g) for 30 min. The supernatant wascollected, aliquoted, and stored at −70° C. until use. Generally, thecrude extract was thawed at 37° C. prior to chromatographicpurification.

Example 3. DEAE-Sepharose FF Chromatography

DEAE-Sepharose FF was used in this Example of the purification methodsfor CBS because it possesses good capacity and flow properties and hasbeen manufactured consistently for several years. This step employed adrip/gravity column that contained approximately 6 mL of resin. Thecolumn was equilibrated in Sodium Phosphate buffer with 50 mM NaCl, pH7.0. Loading of the crude extract was targeted at approximately 20 mgtotal protein/mL resin. After loading the column, the red color of theload was concentrated near the top of the column. Following a wash withequilibration buffer, the column was washed with a buffer containing 150mm NaCl, whereby the majority of color eluted from the column (all stepswere performed at pH 7.0). Essentially all color was removed from thecolumn with a 300 mM NaCl wash. Based on these results, a column waspacked that could be operated in flow mode. The conditions employedequilibration/loading at a NaCl concentration 50 mM, with elution at 250mM NaCl. The final conditions required dilution of the column load withH₂O to approach the ionic strength of the equilibration/wash buffer (50mM NaCl), and elution with 137 mM NaCl (FIGS. 1, 2 and 3). Samples wereanalyzed by SDS-PAGE to determine the relative amounts of CBS proteinand impurities (FIG. 4). The following tables represent columnoperational parameters and data from the scale-up runs that employedthem.

TABLE 1 Operational Parameters for DEAE Capture Step Column Contact timeColumn load target NaCl Concentration Volumes Column vol./flow rateProcess Step (total protein mg/mL) (with 20 mM Na₃PO₄ pH 7.0) (mL)(min.) Equilibration N/A 50 mM 3-5 10 Load 20-25 mg/mL Approx. 50 mMVariable  15 Wash N/A 50 mM 3 10 Elution N/A 137 mM  Variable* 15 2MNaCl Strip N/A 2M 3 10

Note:

Eluate collection started at approximately 0.4 AU and ended atapproximately 0.55 AU. Void volume was typically approximately 1 columnvolume.

TABLE 2 Data from Scale-up Runs (n = 6) Input Column loading (per mLResin) Output Total Protein Recovery Fold Purif. (mg) Units (%) (ByS.A.) 14.5-19.8 3275-5443 79.3-93.0 2.5-3.3 Range 18.2 4451 86 2.8Average

Example 4. IMAC Chromatography

The ability for an immobilized metal affinity column (IMAC) to separateCBS protein from impurities and other contaminants from a biologicalsource, such as a recombinant bacterial cell homogenate, wasdemonstrated. Because of the desire to avoid low pH conditions (<5,anecdotal), varying concentrations of imidazole were used to modulatepartitioning during the chromatography.

Copper (Cu) was tested as a candidate species of IMAC column based onits relatively strong binding characteristics. Prior to being applied tothe IMAC column, the CBS solution was adjusted to 0.4M NaCl. The resultsindicated that capture was near complete, with an acceptable activityrecovery (70-80%). Recovery of CBS was obtained using 100 mM imidazole,which resulted in significant precipitation upon thawing from storage at−70° C. (FIG. 11). In addition, there was only a small increase inpurity relative to the load. Thus, experiments employing Ni′IMAC wereconducted as the metal of choice. In these experiments, the CBS samplewas run through a G-25 column to remove dithiothreitol (DTT) prior toloading the solution onto the IMAC column. Purity enhancement remainedlow and selectivity was similar to Cu⁺⁺, as evidenced by a relativelysmall A₂₈₀ peak in the high imidazole strip fraction. (FIGS. 8, 9 and10).

The relatively weak binding Zn′ was also tested. Although capture, washand elution conditions required significantly lower imidazoleconcentrations, potential for purity enhancement provided positiveresults due to the significant size of the A₂₈₀ peaks in the post-loadwash and high imidazole strip fractions. 0.4 M NaCl and 0.01% TritonX-100 were added to the equilibration and wash buffers to minimizenon-specific binding. (FIGS. 1 and 3). Samples were analyzed by SDS-PAGEto determine the relative amounts of CBS protein and impurities (FIG.13). The results of the IMAC experiments are presented in FIG. 7. Thefollowing tables represent column operational parameters and data fromthe scale-up runs that employed them.

TABLE 3 Operational Parameters for Zn-IMAC Step Contact time ProcessColumn load target Imidazole Concentration Column Volumes Columnvol./flow rate Step (total protein mg/mL) (with 20 mM Na₃PO₄ pH 7.0)(mL) (min.) Equilibration N/A 1 mM 3 10 Load 10 0 Variable  10 Wash N/A1 mM 3 10 Elution N/A 11 mM  Variable* 10 Strip N/A 100 mM  3 10

Note:

Eluate collection started at approximately 0.25 AU and ended atapproximately 0.16 AU. Void volume was typically approximately 1.5column volumes.

TABLE 4 Data from Scale-up Runs (n = 5) Input Column loading (per mLResin) Output Total Protein Recovery Fold Purif. (mg) Units (%) (ByS.A.) 6.5-9.3 4414-7038 71.8-84.6 1.3-1.6 Range 8.1 5687 80 1.4 Average

Example 5. HIC Chromatography

Multiple experiments were conducted to identify the parameters for HICchromatography. Initial drip column experiments were conducted thatemployed a resin with a relatively strong binding ligand (phenyl) withan IMAC eluate as starting material/load. This experiment resulted inempirically complete binding at 1.3M (NH₄)₂SO₄. However, there wasevidence of significant retention of CBS on the column even afterwashing with a low ionic strength buffer. Based on these results, aresin with a weaker binding ligand (butyl) was tested. Initialexperiments with this resin showed no apparent capture at 0.5M(NH₄)₂SO₄. The non-binding flow through of this column experiment wascollected and adjusted to 1.25M (NH₄)₂SO₄, and reloaded on to a columnequilibrated to the same concentration of (NH₄)₂SO₄. In this case therewas evidence of significant binding to the column. A 20 column volume(NH₄)₂SO₄ gradient elution was performed from 1.25M to 0.25M (NH₄)₂SO₄with fractions collected. SDS-PAGE analysis of the fractions indicatedthat there was significant potential for impurity clearance on the lowerend of the gradient. Experiments utilizing step gradient washes atvarying concentrations of (NH₄)₂SO₄ determined the final operationalparameters. (FIGS. 1 and 3). Those parameters and the scale-up run dataare summarized in the tables below.

TABLE 5 Operational parameters for HIC Step (n = 6) Column Contact timeProcess Column load target (NH₄)₂SO₄ Concentration Volumes Columnvol./flow rate Step (total protein mg/mL) (with 20 mM Na₃PO₄ pH 7.0)(mL) (min.) Equilibration N/A 1.4M 3 10 Load 5-10 1.4M Variable  10 WashN/A 1.4M 3 10 Elution N/A 1.1M Variable* 10 Strip N/A 0.05M NaCl 3 10

Note:

Eluate collection starts at approx. 0.25 AU and ends at approx. 0.15 AU.Void volume typically approx. 1.4 column volumes.

TABLE 6 Data from Scale-up Runs (n = 5) Input Column loading (per mLResin) Output Total Protein Recovery Fold Purif. (mg) Units (%) (ByS.A.) 5.1-7.2 5375-9248 77.8-92.7 1.0-1.3 Range 6.3 7638 85 1.2 Average

Example 6. CHAP Chromatography

Ceramic hydroxyapatite is a resin that has a unique, potentially mixedbinding mode chemistry that was utilized in a CBS purification method.CBS displayed acidic characteristics and therefore initial investigationfocused on using phosphate-modulated partitioning. The initialexperiments utilized HIC eluate that was buffer exchanged into a 0.05MNaCl, 0.005M Potassium Phosphate (pH 6.8) buffer. A 5 mL ceramichydroxyapatite (Type 1) cartridge was equilibrated in the same bufferand the conditioned HIC eluate was loaded onto the column. There was novisible breakthrough of protein (as measured by A₂₈₀) during the loadand subsequent wash with equilibration/wash buffer. A linear gradient(5%) of 0.005M to 0.5M Potassium Phosphate was then run and fractionswere collected. Based on the chromatogram, samples were analyzed bySDS-PAGE to determine the relative amounts of CBS protein andimpurities. In subsequent experiments (based on analysis of the resultsof previous experiments), step washes with varying levels of phosphatewere employed to determine optimal conditions for load, wash, andelution steps. In addition, the composition of buffer salts wastransitioned from Potassium to Sodium Phosphate. (FIG. 3). The followingtables represent column operational parameters and data from thescale-up runs that employed them.

TABLE 7 Operational Parameters for CHAP Step Contact time Column loadtarget Na₃PO₄ Concentration Column Volumes Column vol./flow rate ProcessStep (total protein mg/mL) (with 50 mM NaCl, pH 7.0) (mL) (min.)Equilibration N/A 10 mM 3 6 Load 10-15 10 mM Variable  6 Wash N/A 30 mM3 6 Elution N/A 90 mM Variable* 6 Strip N/A 150 mM  3 6

Note: Eluate collection started at approximately 0.20 AU and ends atapproximately 0.16 AU. Void volume was typically approximately 1.0column volumes.

TABLE 8 Data from Scale-up Runs (n = 5) Input Column loading (per mLResin) Output Total Protein Recovery Fold Purif. (mg) Units (%) (ByS.A.) 9.9-12.2 11205-12297 84.6-92.4 1.1-1.2 Range 11.1 11751 89 1.2Average

Example 7. Integrated Process Results

The particular multi-step method described in these Examples wasevaluated at the scale of a 60 mL capture column. All of thepurification trains utilized starting material (crude extract) obtainedfrom fermentations that were seeded with recombinant cells comprising aconstruct comprising a truncated variant of human CBS encoded by anucleic acid having codons optimized for expression in E. coli. Thisconstruct resulted in starting material that was approximately 2-foldhigher in specific activity, and significantly impacted the final purityachieved from the integrated purification method. The overallpurification results using the multi-step method were measured bySDS-PAGE and Specific Activity (FIGS. 5 and 6). The results demonstratedthat the purity and specific activity met or exceeded that of thepurified tagged truncated CBS. All Specific Activities of final columneluates obtained by the largest scale currently possible exceeded 1200U/mg total protein. The following table summarizes the overallpurification results from the scale-up runs.

TABLE 9 Overall Results from Scale-Up Runs Total Recovery (%) FoldPurification Range Average Range Average 3 Column Train (n = 3) 57-60 585.7-6.2 5.9 4 Column Train (n = 2) 47-52 50 4.6-5.4 5.0

Specific Activity of Final Column Eluate=1206-1509.

Having described the invention in detail and by reference to specificembodiments thereof, it will be apparent that modifications andvariations are possible without departing from the scope of theinvention defined in the appended claims. More specifically, althoughsome aspects of the present invention are identified herein asparticularly advantageous, it is contemplated that the present inventionis not necessarily limited to these particular aspects of the invention.

TABLE 10 CβS Sequences Molecule SEQ ID NO Sequence Native human CβS 1atgccttctgagaccccccaggcagaagtggggcccacaggctgccccca nucleic acid sequenceccgctcagggccacactcggcgaaggggagcctggagaaggggtccccagaggataaggaagccaaggagcccctgtggatccggcccgatgctccgagcaggtgcacctggcagctgggccggcctgcctccgagtccccacatcaccacactgccccggcaaaatctccaaaaatcttgccagatattctgaagaaaatcggggacacccctatggtcagaatcaacaagattgggaagaagttcggcctgaagtgtgagctcttggccaagtgtgagttcttcaacgcgggcgggagcgtgaaggaccgcatcagcctgcggatgattgaggatgctgagcgcgacgggacgctgaagcccggggacacgattatcgagccgacatccgggaacaccgggatcgggctggccctggctgcggcagtgaggggctatcgctgcatcatcgtgatgccagagaagatgagctccgagaaggtggacgtgctgcgggcactgggggctgagattgtgaggacgcccaccaatgccaggttcgactccccggagtcacacgtgggggtggcctggcggctgaagaacgaaatccccaattctcacatcctagaccagtaccgcaacgccagcaaccccctggctcactacgacaccaccgctgatgagatcctgcagcagtgtgatgggaagctggacatgctggtggcttcagtgggcacgggcggcaccatcacgggcattgccaggaagctgaaggagaagtgtcctggatgcaggatcattggggtggatcccgaagggtccatcctcgcagagccggaggagctgaaccagacggagcagacaacctacgaggtggaagggatcggctacgacttcatccccacggtgctggacaggacggtggtggacaagtggttcaagagcaacgatgaggaggcgttcacctttgcccgcatgctgatcgcgcaagaggggctgctgtgcggtggcagtgctggcagcacggtggcggtggccgtgaaggctgcgcaggagctgcaggagggccagcgctgcgtggtcattctgcccgactcagtgcggaactacatgaccaagttcctgagcgacaggtggatgctgcagaagggctttctgaaggaggaggacctcacggagaagaagccctggtggtggcacctccgtgttcaggagctgggcctgtcagccccgctgaccgtgctcccgaccatcacctgtgggcacaccatcgagatcctccgggagaagggcttcgaccaggcgcccgtggtggatgaggcgggggtaatcctgggaatggtgacgcttgggaacatgctctcgtccctgcttgccgggaaggtgcagccgtcagaccaagttggcaaagtcatctacaagcagttcaaacagatccgcctcacggacacgctgggcaggctctcgcacatcctggagatggaccacttcgccctggtggtgcacgagcagatccagtaccacagcaccgggaagtccagtcagcggcagatggtgttcggggtggtcaccgccattgacttgctgaacttcgtggccgcccaggagcgggaccag aagtgaNative human CβS 2 MPSETPQAEVGPTGCPHRSGPHSAKGSLEKGSPEDKEAKEPLWIRPDAPSpolypeptide sequence RCTWQLGRPASESPHHHTAPAKSPKILPDILKKIGDTPMVRINKIGKKFGLKCELLAKCEFFNAGGSVKDRISLRMIEDAERDGTLKPGDTIIEPTSGNTGIGLALAAAVRGYRCIIVMPEKMSSEKVDVLRALGAEIVRTPTNAREDSPESHVGVAWRLKNEIPNSHILDQYRNASNPLAHYDTTADEILQQCDGKLDMLVASVGTGGTITGIARKLKEKCPGCRIIGVDPEGSILAEPEELNQTEQTTYEVEGIGYDFIPTVLDRIVVDKWEKSNDEEAFTFARMLIAQEGLLCGGSAGSTVAVAVKAAQELQEGQRCVVILPDSVRNYMTKFLSDRWMLQKGFLKEEDLTEKKPWWWHLRVQELGLSAPLTVLPTITCGHTIEILREKGFDQAPVVDEAGVILGMVTLGNMLSSLLAGKVQPSDQVGKVIYKQFKQIRLTDTLGRLSHILEMDHFALVVHEQIQYHSTGKSSQRQMVEGVVTAIDLLNEVAAQERDQ K Truncated, Human 3MPSETPQAEVGPTGCPHRSGPHSAKGSLEKGSPEDKEAKE CβS polypeptidePLWIRPDAPSRCTWQLGRPASESPHHHTAPAKSPKILPDI sequenceLKKIGDTPMVRINKIGKKFGLKCELLAKCEFFNAGGSVKDRISLRMIEDAERDGTLKPGDTIIEPTSGNTGIGLALAAAVRGYRCIIVMPEKMSSEKVDVLRALGAEIVRTPTNARFDSPESHVGVAWRLKNEIPNSHILDQYRNASNPLAHYDTTADEILQQCDGKLDMLVASVGTGGTITGIARKLKEKCPGCRIIGVDPEGSILAEPEELNQTEQTTYEVEGIGYDFIPTVLDRTVVDKWFKSNDEEAFTFARMLIAQEGLLCGGSAGSTVAVAVKAAQELQEGQRCVVILPDSVRNYMTKFLSDRWMLQKGFLKEE DLTEKKPWWWHLRTruncated, Optimized 4 ATGCCGTCAGAAACCCCGCAGGCAGAAGTGGGTCCGACGGHuman CβS nucleic GTTGCCCGCACCGTAGCGGTCCGCATTCTGCAAAAGGCAG acid sequenceTCTGGAAAAAGGTTCCCCGGAAGATAAAGAAGCCAAAGAACCGCTGTGGATTCGTCCGGACGCACCGTCACGCTGTACCTGGCAGCTGGGTCGTCCGGCAAGCGAATCTCCGCATCACCATACGGCTCCGGCGAAAAGTCCGAAAATTCTGCCGGATATCCTGAAGAAAATTGGTGACACCCCGATGGTTCGTATCAACAAAATCGGCAAAAAATTCGGTCTGAAATGCGAACTGCTGGCTAAATGTGAATTTTTCAATGCGGGCGGTTCCGTGAAAGATCGTATCTCACTGCGCATGATTGAAGATGCTGAACGCGACGGCACCCTGAAACCGGGTGATACGATTATCGAACCGACCTCTGGCAACACGGGTATCGGTCTGGCACTGGCGGCGGCAGTCCGTGGTTATCGCTGCATTATCGTGATGCCGGAAAAAATGAGCTCTGAAAAAGTTGATGTCCTGCGTGCTCTGGGCGCGGAAATTGTTCGTACCCCGACGAATGCCCGCTTCGACAGTCCGGAATCCCATGTGGGTGTTGCATGGCGCCTGAAAAACGAAATCCCGAATTCGCACATTCTGGATCAGTATCGTAACGCTAGCAATCCGCTGGCGCATTACGATACCACGGCCGACGAAATCCTGCAGCAATGTGATGGCAAACTGGACATGCTGGTCGCTTCTGTGGGTACCGGCGGTACCATTACGGGCATCGCGCGTAAACTGAAAGAAAAATGCCCGGGCTGTCGCATTATCGGTGTGGATCCGGAAGGCAGTATTCTGGCGGAACCGGAAGAACTGAACCAGACCGAACAAACCACGTATGAAGTTGAAGGCATCGGTTACGATTTTATTCCGACCGTCCTGGATCGCACGGTGGTTGACAAATGGTTCAAAAGCAATGACGAAGAAGCCTTTACCTTCGCACGTATGCTGATCGCTCAGGAAGGTCTGCTGTGCGGTGGTTCAGCAGGTTCGACGGTCGCAGTGGCAGTTAAAGCTGCGCAGGAACTGCAAGAAGGTCAACGTTGTGTCGTGATTCTGCCGGATTCTGTTCGCAACTACATGACCAAATTTCTGAGTGACCGTTGGATGCTGCAAAAAGGCTTCCTGAAAGAAGAAGATCTGACCGAGAAAAAACCGTGGTGGTGGCACCTGCGCT AA

1. A method for purifying cystathionine β-synthase (CBS) proteincomprising: (a) providing a CBS-containing solution comprising one or aplurality of impurities; and (b) performing chromatographic separationof the CBS-containing solution using an ion exchange chromatographycolumn; (c) performing chromatographic separation of the CBS-containingsolution using a metal affinity chromatography (IMAC) resin; (d)performing chromatographic separation of the CBS-containing solutionusing a ceramic hydroxyapatite resin; and (e) performing chromatographicseparation of the CBS-containing solution using a hydrophobicinteraction chromatography (HIC) column; wherein the CBS protein is theCBS protein of SEQ ID NO: 2 or a truncation thereof.
 2. The method ofclaim 1, wherein the ion exchange chromatography column is a weak anionexchanger.
 3. The method of claim 2, wherein the weak anion exchanger isa DEAE-Sepharose FF column, a DEAE-Sephacel column, a DEAE-cellulosecolumn, a DEAE-Sephadex column, or a QAE-Sephadex column.
 4. The methodof claim 1, wherein the metal affinity chromatography (IMAC) resin ischarged with a divalent metal ion.
 5. The method of claim 4, wherein thedivalent metal ion is nickel, copper, cobalt, or zinc.
 6. The method ofclaim 4, wherein the first chromatographic separation step is IMACchromatography and the IMAC resin is charged with nickel or copper. 7.The method of claim 4, wherein the first chromatographic separation stepis ion exchange chromatography followed by IMAC chromatography, andwherein the IMAC resin is charged with zinc.
 8. The method of claim 1,further comprising eluting the CBS protein from the metal affinitychromatography (IMAC) resin with an elution buffer comprising imidazole.9. The method of claim 1, wherein the CBS protein is a chemicallycleaved or genetically engineered carboxyl truncated CBS protein havinga carboxyl-terminal truncation resulting in an ending position of one ofamino acid residues from 382-550 of SEQ ID NO:
 2. 10. The method ofclaim 1, wherein the CBS protein has the amino acid sequence of SEQ IDNO:
 3. 11. The method of claim 1, wherein the CBS-containing solution isa clarified CBS solution.
 12. The method of claim 1, wherein the CBSprotein is produced in a recombinant cell.
 13. The method of claim 12,wherein the CBS-containing solution is obtained by homogenizingrecombinant cells expressing a recombinant construct comprising anucleic acid sequence encoding the CBS protein.
 14. The method of claim13, wherein homogenizing is performed by disrupting the cells usingsonication.
 15. The method of claim 13, wherein homogenizing isperformed by disrupting the cells by exposing the cells to a lysozyme.16. The method of claim 12, wherein the nucleic acid sequence encodingthe CBS protein comprises SEQ ID NO:
 4. 17. The method of claim 1,wherein the CBS protein binds to a chromatography matrix duringchromatographic separation.
 18. The method of claim 17, furthercomprising eluting the CBS protein from the chromatography matrix toobtain a substantially pure CBS protein.
 19. A method of preparing apharmaceutical composition comprising combining a substantially purifiedCBS solution prepared according to the method of claim 1 and apharmaceutically acceptable carrier.